Multigene Sequence-Based and Phenotypic Characterization Reveals the Occurrence of a Novel Entomopathogenic Nematode Species, Steinernema anantnagense n. sp.

Abstract Three entomopathogenic nematode populations were isolated from agricultural fields in the Anantnag district of Jammu and Kashmir (India). Sequences of multiple gene regions and phenotypic features show that they are conspecific and represent a novel species. Molecular and morphological features provided evidence for placing the new species into the “Kushidai” clade. Within this clade, analysis of sequence data of the internal transcribed spacer (ITS) gene, the D2D3 region of the 28S rRNA gene, the mitochondrial cytochrome oxidase I (mtCOI) gene, and the mitochondrial 12S (mt12S) gene depicted the novel species as a distinctive entity closely related to Steinernema akhursti, S. kushidai, and S. populi. Phylogenetic analyses also show that the new species is a sister species to S. akhursti, and these two species are closely related to S. kushidai and S. populi. Additionally, the new species does not mate or produce fertile progeny with any of the closely related species, reinforcing its uniqueness from a biological species concept standpoint. The new species is further characterized by the third-stage infective juveniles with almost straight bodies (0.7–0.8 mm length), poorly developed stoma and pharynx, and conoid-elongate tail (49–66 µm) with hyaline posterior part. Adult females are characterized by short and conoid tails bearing a short mucron in the first generation and long conoid tails with thin mucron in the second generation. Adult males have ventrally curved spicules in both generations. Moreover, the first-generation male has rounded manubrium, fusiform gubernaculum, conoid and slightly ventrally curved tails with minute mucron, and the second generation has rhomboid manubrium anteriorly ventrad bent, and tails with long and robust mucron. The morphological, morphometrical, molecular, and phylogenetic analyses support the new species status of this nematode, which is hereby described as Steinernema anantnagense n. sp. The bacterial symbiont associated with S. anantnagense n. sp. represents a novel species, closely related to Xenorhabdus japonica. These findings shed light on the diversity of entomopathogenic nematodes and their symbiotic bacteria, providing valuable information for future studies in this field.

Entomopathogenic nematodes (EPNs) of the genus Steinernema (Travassos, 1927) are among the most important biological control agents used in agriculture to control insect pests. The nematodes of this genus are associated with entomopathogenic bacteria of the genus Xenorhabdus, carried in a specialized receptacle structure hosted in the digestive tract of the free-living infective juveniles (IJs) (Chaston et al., 2011). The infective juveniles search for insects, and once inside the hosts, they release their bacterial symbiont into the hemocoel. Bacteria kill the insect hosts via toxins, enzymes, and insecticidal compounds produced during bacteria multiplication, making these symbiotic organisms highly valuable pest management tools in sustainable and ecofriendly agriculture.
In order to characterize the prevalence and distribution of EPNs in Indian soils, a survey was conducted in the Pir Panjal Range, in the Kashmir region of the Indian subcontinent. As a result of this survey, several nematode populations were recovered, including three isolates: Steiner_6, Steiner_7, and Steiner_8. Initial molecular characterization suggests that these three isolates are conspecific and represent a new species in the genus Steinernema. In this study, we describe Steinernema anantnagense n. sp. based on morphological observations and morphometric analysis using light microscopy (LM) and scanning electron microscopy (SEM), as well as molecular studies based on genetic sequences of ribosomal RNA and mitochondrial genes. Self-crossing and cross-hybridization experiments were also used. In addition, we isolated and characterized the symbiotic bacterium associated with S. anantnagense n. sp.

Nematode survey and collection
Steinernema anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 nematodes were isolated from soil samples collected in the Pir Panjal Range of Kashmir Valley, India using Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) larvae as a bait insect. The isolates Steiner_6, Steiner_7, and Steiner_8 were collected in the Waghama area of Bijbehara Anantnag of the union territory of Jammu and Kashmir (GPS coordinates: 33.828914, 75.100091; 1606 m above the sea level) from soils around roots of willow, walnut, and apple intercrops, respectively, in areas adjoining district Anantnag, India. The insect cadavers recovered from soil samples were washed with ddH 2 O, sterilized with 0.1% NaOCl 2 , and nematode IJs were recovered from them by the White trap method (White, 1927). The IJs were sterilized with 0.1% NaOCl 2 and stored in 250 mL tissue culture flasks in Biological Oxygen Demand incubator at 15°C. The new species has been registered in the ZooBank at urn:lsid:zoobank.org:pub:210D5242-2C15-437F-8D57-B00EECD98B85.

Morphological and morphometrical characterization
Different life stages of S. anantnagense n. sp. were obtained from infected Galleria mellonella larvae exposed to 100 IJs/insects in a 15 cm-diameter Petri dish lined with moistened Whatman number 1 filter paper and kept in the dark at 25°C. The wax moth larvae died within 48 h after inoculation. After they died, the insect cadavers were transferred to a modified White trap (Kaya & Stock, 1997) and incubated at 25°C until IJs emerged. First-and second-generation adult nematodes were obtained by dissecting infected G. mellonella cadavers in Ringer's solution after 3-4 and 6-7 days of infection, respectively. Infective juveniles (IJs) were collected after they emerged from G. mellonella cadavers in White traps (White, 1927). Nematodes were killed with water at 60°C, fixed in 4% formalin solution (4 mL formaldehyde, 1 mL Glycerol, 95 mL ddH2O), dehydrated by the Seinhorst method (Seinhorst, 1959), and transferred to anhydrous glycerin. Nematodes were, after that, picked with a peacock feather and mounted on permanent glass slides with extra layers of paraffin wax to prevent the flattening of the nematodes as described (Bhat et al., 2022). Morphometric measurements were taken using the Nikon DS-L1 image acquisition software mounted on a phase-contrast microscope (Nikon Eclipse 50i) in μm. Light microscopy photographs were captured using a Nikon Eclipse 80i microscope (Olympus, Tokyo, Japan) equipped with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera. For the scanning electron microscopy (SEM), nematodes preserved in 4% formalin were re-hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried with liquid CO 2 , mounted on SEM stubs with a carbon tape, coated with gold in sputter coater, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany) (Abolafia, 2015). All micrographs were processed using Adobe® Photoshop® CS. Morphological characters of closely related species were taken from the original publications. The terminology used for the morphology of stoma and spicules follows the proposals by De Ley et al. (1995) and Abolafia and Peña-Santiago (2017a), respectively, and the terminology for pharynx follows the proposals by Bird and Bird (1991) and Baldwin and Perry (2004).

Self-crossing and cross-hybridization experiments
Self-crossing and cross-hybridization experiments were carried out using G. mellonella larvae hemolymph as described by Kaya & Stock, 1997 with minor modifications. To this end, drops of hemolymph obtained from surface-sterilized G. mellonella larvae were placed in sterile Petri dishes (35×10 mm). Hemolymph drops were treated with a small amount of phenylthiourea to prevent melanization. Then 40-60 surface-sterilized IJs (0.1% NaOCl for 30 min, followed by thrice rinse through sterile distilled water) were added to the hemolymph drops. Then, Petri dishes were wrapped in moistened tissue paper and kept in plastic bags at 25°C (room temperature). Petri dishes were observed daily for the presence of males and virgin females. Then, males and virgin females in the ratio of 3:3 were placed separately in fresh hemolymph drops and were crossed with adults of the opposite sex of the other species. Controls consist of crosses of identical isolates; some females were kept without males to check their virginity (n=30). The Petri dishes were observed daily for 15 days to determine the production of offspring. Experiments were conducted twice under the same conditions. The following species were crossed: Steinernema anantnagense n. sp. (Steiner_6, Steiner_7, and Steiner_8), S. ichnusae Sardinia, S. litorale Aichi, S. weiseri, S. akhursti Akh, S. citrae, S. cholashanense GARZE, S. feltiae P1, S. silvaticum, S. africanum RW14-M-C2a-3, and S. xueshanense DEQ.
To obtain genomic sequences of nematodes that belong to all the validly described species closely related to S. anantnagense n. sp., we searched the database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). Steinernema monticola (AB698756, GU395647, AY943994, and AY944020) was used as an outgroup in ITS, D2D3, mtCOI, and mt12S based phylogenetic trees. The resulting sequences were aligned with MUSCLE (v3.8.31) (Edgar, 2004) and used to reconstruct phylogenetic relationships by the Maximum Likelihood method based on the following nucleotide substitution models: Hasegawa-Kishino-Yano model (HKY+G) (ITS), Tamura-Nei (TN93+G+I) (D2-D3 & COI), and Tamura 3-parameter (T92+G) (12S). To select the best substitution models, bestfit nucleotide substitution model analyses were conducted in MEGA 11 (Nei & Kumar, 2000;Tamura et al., 2021). The trees with the highest log likelihood are shown. The percentages of trees where the associated taxa clustered together are displayed next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. In some cases, a discrete Gamma distribution (+G) was used to model evolutionary rate differences among sites and the rate variation model allowed for some sites to be evolutionarily invariable (+I). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Graphical representation and edition of the phylogenetic trees were performed with Interactive Tree of Life (v3.5.1) (Chevenet et al., 2006;Letunic and Bork, 2016).

Symbiotic relationships
The Xenorhabdus entomopathogenic bacteria associated with S. anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 nematodes were isolated as described previously (Machado et al., 2018;Machado et al., 2019). Briefly, G. mellonella (n = 10) larvae were exposed to 100 nematode infective juveniles. Two to three days later, insect cadavers were surfacesterilized with 0.1% sodium hypochlorite solution and cut open with a sharp blade. Sterile polypropylene inoculation loops were inserted into the cadaver, and the loops were then streaked on LB agar plates and incubated at 28°C for 24-48 h. Xenorhabduslike colonies were sub-cultured until monocultures were obtained. The strains were further sub-cultured and maintained on LB agar plates at 28°C. To establish their taxonomic identities, we reconstructed phylogenetic relationships based on whole genome sequences of the isolated bacteria and all the different species of the genus Xenorhabdus  and genomic sequences were obtained as described by Machado et al. (2021). Genome sequences were deposited in the National Centre for Biotechnology Information, and accession numbers are listed in Table S3. Phylogenetic relationships were reconstructed based on the assembled genomes and the genome sequences of all validly published species of the genus with publicly available genome sequences as described by Machado et al. (2023). Whole genome sequence similarities were calculated by the digital DNA-DNA hybridization (dDDH) method using the recommended formula 2 of the genome-togenome distance calculator (GGDC) web service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Auch et al., 2010(Auch et al., , 2020Meier-Kolthoff et al., 2013, 2014.

Results and Discussion
Three populations of Steinernema nematodes, Steiner_6, Steiner_7 and, Steiner_8, were isolated from agricultural soils in the Bijbehara area of district Anantnag, India. Analysis of several taxonomically relevant markers show that Steiner_6, Steiner_7, and Steiner_8 are conspecific, belong to the "Feltiae-Kushidai-Monticola" superclade or "Kushidai" clade, are closely related to S. akhursti, S. populi, S. sangi, and S. kushidai, and represent a new species, for which we propose the name Steinernema anantnagense n. sp. To describe this new species, we compared this species with other closely related species at the molecular and morphological levels and conducted cross-hybridization and self-crossing experiments. As all three populations are identical at the molecular level, we selected Steiner_7 for in-depth morphological and morphometrical identification.

First generation females (n = 20)
Body 2.3-4.9 mm long, and C-shaped after heat relaxation and fixation. Cuticle with poorly visible annuli, with fine transversal incisures. Lateral fields absent. Labial region rounded, and continuous with the adjacent part of the body. Labial plate with six lips that are fused together, each with one labial papilla at the tip and one lower cephalic papilla each except for the lateral lips. Amphid openings present at the lateral lips, close to the labial papilla, with a small transversal slit. Stoma funnel-shaped, shallow, short, and wider at the anterior part. Cheilostom short with rounded and refringent rhabdia; gymnostom scarcely developed with a minute rhabdia; stegostom robust, slightly wider than long, with a funnel-shaped lumen and walls with very minute rhabdia. Pharynx muscular with a subcylindrical procorpus, a somewhat swollen metacorpus, a short and robust isthmus, and a spheroid basal bulb with reduced valves. Nerve ring surrounds the posterior part of the isthmus. Secretory-excretory pore circular, located at the anterior part of the isthmus. Deirids inconspicuous. Cardia short, conoid, and surrounded by intestinal tissue. Intestine tubular without differentiation, with thinner walls at the anterior end. Reproductive system didelphic, amphidelphic, and ovaries are reflexed in dorsal position. Oviducts well developed with glandular spermatheca, and uteri tubular with numerous uterine eggs. Vagina short with muscular walls, vulva protruding in the form of a transverse slit. Rectum 0.3-0.6 times the anal body diameter, with three small rectal glands. Anus well developed. Tail conoid, shorter than body anal diameter, with an acute terminus. Phasmids located at the posterior part of the tail, at 25-30% of the tail length.

Second generation females (n = 20)
Similar to first generation females, but smaller, measuring 1.8-2.4 mm in length. Tail conoid, with an acute terminus, longer than the first generation females.

First generation males (n = 20)
Body 1.2-1.9 mm long, ventrally curved posteriorly, C-or J-shaped when heat killed. General morphology similar to that of females. Reproductive system monorchic, with the testis ventrally reflexed. Spicules paired, symmetrical, ventrally curved with a welldeveloped manubrium, either rounded or spoonshaped. Calamus short and narrower, lamina ventrad curved at the anterior part and bears longitudinal ribs, ending in a blunt terminus. Velum indistinct, does not reach the spicule tip, and with no rostrum or retinaculum. Gubernaculum with a rounded manubrium, a fusiform corpus and a narrower and elongated tip, 0.5-0.7 times spicules length. Tail conoid with a rounded terminus bearing a fine mucron. Bursa absent. 11 pairs of genital papillae and a single mid-ventral papilla present, arranged as follows: five pairs (GP1-GP5) subventral precloacal, one pair (GP6) lateral precloacal, one single (MP) midventral precloacal, two pairs (GP7-GP8) sub-ventral        Second generation males (n = 20) Morphology of second generation males similar to that of the first generation males, but smaller, 0.8-1.2 mm in length. Tail with long, straight and robust mucron. Spicules curved ventrally, with a rhomboid shaped manubrium, slightly broader than the calamus, and a lamina curved ventrally at the anterior part. Ventral velum very reduced, and two longitudinal lateral ribs present. Gubernaculum with a slightly ventrad curved manubrium that is rounded and ventrad bent, slightly fusiform corpus and a narrower and slender terminus. Arrangement of genital papillae and phasmids similar to that of first generation males.
Infective juvenile (L3 stage) (n = 20) IJ body 0.7-0.8 mm long, almost straight or slightly curved body shape when heat-killed. Body tapers gradually at both extremes, cuticle with transverse incisures, well-developed annuli. Lateral fields begin as a single line close to the anterior end, and increase to eight ridges before gradually reducing to five and then two near the anus and phasmid levels, respectively. Lip region slightly narrower than the adjacent part of the body, six amalgamated lips; with smaller lateral lips, six reduced labial and four prominent cephalic papillae. Amphidial apertures pore-like, oral opening triangular with a noticeable margin. Stoma reduced and tubular with a small lumen, consisting of a short cheilostom and an elongated gymno-stegostom. Pharynx elongated and narrow, with a very long corpus; a slightly narrower isthmus, and a pyriform basal bulb with reduced valves. Nerve ring surrounds the isthmus, excretory pore located at the metacorpus level. Hemizonid present. Deirids inconspicuous. Cardia conoid. Intestine bears a bacterial sac at the anterior part. Rectum long, almost straight, with very short cuticular and elongated cellular parts, anus distinct. Genital primordium located at the equatorial region, tail conoid, tapering gradually to a pointed terminus, with a longer cellular part than the hyaline part and an irregular cellular-hyaline junction. Phasmids located at 37-45% of the tail length.

Life cycle
Steinernema anantnagense n. sp. is a highly pathogenic nematode species that can be easily reared on G. mellonella larvae at a temperature ranging from 18-24°C. The life cycle of this new species is similar to the life cycle of other Steinernema species. When G. mellonella larvae are exposed to 50-100 infective juveniles (IJs), they die within 24-48 h. The first-and second-generation adults of S. anantnagense n. sp. can be found in the insect cadavers 3-4 and 5-6 days after infection, respectively. The pre-infective juveniles leave the host body, mature for a few days, and then migrate to the water traps after 10-15 days.

Etymology
The species name is derived from the location Anantnag, a District in the Union Territory of Jammu and Kashmir, India, where the nematode specimens used in this study to describe the new species were obtained.

Cross-hybridization experiments
Mating experiments were carried out to determine the reproductive isolation of S. anantnagense n. sp. by pairing males and females of this species with individuals from other Steinernema species, including S. ichnusae, S. litorale, S. weiseri, S. akhursti, S. citrae, S. cholashanense, S. feltiae, S. silvaticum, S. africanum, and S. xueshanense. No offspring were produced when S. anantnagense n. sp. nematodes were allowed to interact with nematodes of the any of the above mentioned species, indicating that S. anantnagense n. sp. is reproductively isolated. Cross tests were also conducted between males and females of Steiner_6, Steiner_7, and Steiner_8 to determine their conspecific status. The results showed that fertile offspring were produced, confirming that they belong to the same species. Controls were also carried out, which included selfcrossed species, and offspring were observed in all of them. However, no progeny were observed in the single-female control plates.
Diagnosis and relationships of Steinernema anantnagense n. sp.
Steinernema anantnagense n. sp. is characterized by adults with a short stoma, rounded cheilorhabdia, and a robust pharynx with a round basal bulb. Females of the first generation are between 2.3-4.9 mm in length, with didelphic-amphidelphic reproductive system, and possess a shorter conoid tail bearing a short mucron in the first generation (c = 61-122, c′ = 0.5-0.8) and a longer conoid tail with thin mucron in the second generation (c = 43-67, c′ = 0.8-1.3). Males are smaller, between 1.2-1.9 mm in length, with a reproductive system that is monorchid and that has ventrally curved spicules bearing rounded manubrium in the first generation and rhomboid manubrium in the second generation, gubernaculum is fusiform in the first generation and anteriorly ventrad bent in the second generation, tail is conoid and slightly ventrally curved with a minute mucron in the first generation (c = 34-64, c′ = 0.8-1.6) and with a longer and more robust mucron in the second generation (c = 31-52, c′ = 0.6-1.4).
Steinernema anantnagense n. sp. belongs to a group of species known as the "Kushidai-clade", and presents several traits common to this group. Several of the morphological and morphometric traits of the IJs and adults overlap with those of other species in the "Kushidai-clade". However, several distinct morphological and morphometrical characteristics can differentiate S. anantnagense n. sp. from these closely related species (Tables 2-4).

Nematode phylogenetic relationships
Phylogenetic reconstructions based on the nucleotide sequences of the internal transcribed spacer (ITS) marker of the rRNA gene, D2D3 expansion segments of the 28S rRNA gene, the cytochrome oxidase subunit I (COI), and the mitochondrial 12S rRNA gene show that S. anantnagense n. sp. Steiner_6, Steiner_7, and Steiner_8 are conspecific and belong to the "Kushidai" clade and the "Feltiae-Kushidai-Monticola" superclade ( Figs. 6 and 7). Phylogenetic analyses of all four abovementioned markers clearly separate S. anantnagense n. sp. from all other species. In addition, these phylogenetic reconstructions show that S. anantnagense n. sp. is closely related to other Asian species, including S. akhursti, S. kushidai, and S. populi. No phylogenetic tree was built using the 18S rRNA genetic region because insufficient 18S rRNA gene sequences are publicly available. However, the resulting sequences were deposited in the NCBI databank under the following accession numbers: OQ407498 (Steiner_6), OQ407499 (Steiner_7), and OQ407500 (Steiner_8).

Symbiotic relationships
Phylogenetic reconstructions based on whole genome sequences show that the bacterial symbiont isolated from S. anantnagense n. sp. Steiner_7, named here XENO-2, is closely related to X. japonica DSM 16522 T and X. vietnamensis VN01 T (Fig. 8). The digital DNA-DNA hybridization (dDDH) values between XENO-2 and X. japonica DSM 16522 T , and between XENO-2 and X. vietnamensis VN01 T are 51.8% and 40.0%, respectively. These values are below the 70% divergence threshold for prokaryotic species delineation, indicating that XENO-2 T represents a novel species within the genus Xenorhabdus (Wayne et al., 1987). This species is formally described elsewhere.   Table S3.
A Side Note on The Nomenclature of Steinernema Monticolum The term "monticolum" was introduced by Stock et al. (1997) to refer to the geographic origin of the nematodes studied, which were collected in Mount Jiri (Sancheong, Gyeongnam province, Korea). This term is a combination of "monti" referring to "mountain" and "colum" derived from the Latin suffix "cola" meaning "that lives in a place." However, it should be noted that, as the suffix "cola" is a masculine noun in Latin, it does not have gender variations. Therefore, the correct term to use is "monticola." The correct usage of this term has been discussed in detail by Nicolson, 1987. In light of this, we propose to refer to this species as Steinernema monticola, as was first used by Choo et al. (1998).

Conclusions
The differences in morphology, morphometry, molecular characteristics, reproductive isolation, and clear phylogenetic distinction support that Steiner_6, Steiner_7, and Steiner_8 represent a new species of entomopathogenic nematodes. We propose to name this species Steinernema anantnagense n. sp. This discovery marks the second new species description in the Steinernema genus from the Indian Subcontinent. Our findings provide valuable insights into the biodiversity and distribution of these biological control agents. Furthermore, our results underscore the importance of accurately characterizing newly described Steinernema species through the inclusion of all three standard rDNA markers (ITS, SSU, and LSU) in combination with the mitochondrial COI gene, in addition to classical taxonomy. We recommend that all future species descriptions follow this approach.

Supplementary Materials
The following tables are available online at https://doi. org/10.2478/jofnem-2023-0029. Table S1: Pairwise distances in base pairs of the mitochondrial cytochrome oxidase subunit I (mt COI) among other Steinernema species and Steinernema anantnagense n. sp. Data for new species are in bold.

Conflicts of Interest
The authors declare no conflict of interest and that no humans or animals were used for experimental purposes. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.